Shear stress acting on endothelial cells produces vasodilation. This is arguably the most important physiological endothelial mechanism of dilation and occurs in virtually every vascular bed. Our recent data indicate that flow-mediated dilation (FMD) occurs in coronary arterioles from patients with coronary disease but operates through a novel mechanism involving endothelial production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2). Surprisingly the mitochondrial respiratory chain plays a necessary role in FMD in the human heart. The overall goal of this application is to examine the FMD signaling sequence from endothelium to smooth muscle studying 3 aims. 1) We will examine the mechanism of endothelial production H2O2. using fresh human coronary arterioles from subjects with coronary disease and cultured human endothelial cells from both microvascular tissue and conduit arteries for comparison. We shall pursue exciting preliminary data that indicate both mitochondria and NADPH oxidase are involved, possibly through a ROS- induced ROS release mechanism and activation of Rac1. 2) Using a novel bioassay technique to assess vasodilation and smooth muscle potassium channel opening, we shall identify the endothelial derived hyperpolarizing factor (EDHF) responsible for dilation. Both arachidonic acid metabolites and H2O2 are necessary for FMD, but preliminary studies point to H2O2 as the transferable dilator agent. 3) We shall determine the mechanism of H2O2 -induced dilation, examining the novel hypothesis that H2O2 directly acts on PKG1 by cysteine oxidation, yielding an activated disulfide dimeric form of the enzyme. These goals span a broad, clinically relevant redox signaling pathway from endothelial H2O2 formation, to H2O2 release as a transferable vasomotor substance, to its mechanism of action on underlying smooth muscle cells. Collectively these aims address a novel mechanism of endothelium-dependent dilation involving mitochondrial generation of ROS, thus far reported only in human hearts. Results should identify new links among cellular mechanotransduction, respiration, and redox signaling that regulate important physiological events such as arteriolar vasodilation, responsible for tissue perfusion. The direct relevance to humans with chronic coronary disease provides a strong foundation for this mechanistic approach to understanding microvascular reactivity.